Arachidonic and linoleic acid derivatives impact oocyte ICSI fertilization--a prospective analysis of follicular fluid and a matched oocyte in a 'one follicle--one retrieved oocyte--one resulting embryo' investigational setting.

OBJECTIVE: To evaluate human oocyte ability to undergo fertilization and subsequent preimplantation embryonic development in relation to a wide panel of follicular fluid (FF) arachidonic acid derivatives (AAD) and linoleic acid derivatives (LAD) of prospectively selected patients undergoing intracytoplasmic sperm injection (ICSI). METHODOLOGY: Study was designed as a two center (a university clinic and a private clinic) prospective study. 54 women of 181 consecutive couples undergoing ICSI were prospectively found to be eligible for analysis. 'One follicle - one retrieved oocyte - one resulting embryo' approach was used. Each individual follicle was aspirated independently and matched to an oocyte growing in this particular follicular milieu. FF samples were assessed for AAD and LAD by high-performance liquid chromatography; additionally, activity of secretory phospholipase A (sPLA2) was determined by enzyme-linked immunosorbent assay. PRINCIPAL FINDINGS: Increased activity of sPLA2 and significantly higher AAD and LAD levels were found in FF of oocytes that did not show two pronuclei or underwent degeneration after ICSI in comparison to oocytes with the appearance of two pronuclei. Receiver operating characteristics curve analysis identified acids with the highest sensitivity and specificity: 5oxo-hydroxyeicosatetraenoic, 16-hydroxyeicosatetraenoic, 9-hydroxyoctadecadieneoic and 12-hydroxyeicosatetraenoic. No significant differences between AAD and LAD related to embryo quality were found. CONCLUSIONS/SIGNIFICANCE: Our study demonstrates for the first time that elevated concentrations of AAD and LAD in FF at the time of oocyte retrieval significantly decrease the ability of oocytes to form pronuclei after ICSI. This may serve as a new tool for non-invasive assessment of oocyte developmental capacity. However, levels of AAD and LAD are not associated with subsequent embryo quality or pregnancy rate, and therefore more studies are needed to determine their usefulness in human IVF procedure.

Effects of substances on serum zinc levels in postmenopausal women.

INTRODUCTION: Demographic facts and forecasts about lengthening life expectancy motivate to systematize the knowledge of health problems experienced by women at the age of 50 and older. It refers to the whole health policy including health economics. Longer female life spans cause that an increasing number of women suffer from health problems associated with the perimenopausal period, and become health care recipients. Also a shift of retirement age is the reason to take interdisciplinary actions for women's health and quality of life. This study describes a decline in the levels of many bioelements in hair, urine and blood serum, which progresses with age. It not only correlates with a decrease in the synthesis and secretion of estrogen, but also environmental pollution, unhealthy lifestyle and the use of substances. AIM OF THE STUDY: The aim of this study was to determine the correlation between serum zinc levels in postmenopausal women and such variables as the use of substances (cigarettes, alcohol) and menopausal hormone therapy (MHT). Material and method: The study was conducted among 152 healthy women being 1-16 years after menopause. The women were divided into study group (MHT users) and control group (MHT non-users). A sub-division criterion was the use of substances (cigarettes, alcohol). Serum zinc levels were determined in all women. Results: The use of substances significantly contributed to the lowering of serum zinc levels in postmenopausal women. MHT users had statistically higher average zinc levels in blood serum, which referred both to smokers and consumers of alcohol and those who did not use these substances. CONCLUSIONS: (1) The use-of substances (cigarettes, alcohol) contributes to the lowering of zinc levels in blood serum. (2) MHT positively affects serum zinc levels in postmenopausal women regardless of whether they use substances (cigarettes, alcohol) or not.

The SLC19A1 80G>A polymorphism is not associated with male infertility.

Previous studies have revealed that genetic factors may be involved in regulating folate turnover, e.g. methylenetetrahydrofolate reductase polymorphism in the development of male infertility. Folate transporter, encoded by the SLC19A1 gene, commonly referred to as reduced folate carrier (RFC) is a transmembrane protein, which transfers hydrophilic folates across the cell membrane. It was hypothesized that common polymorphism within the SLC19A1 gene (rs1051266:G>A, 80G>A) may alter RFC function. The aim of this study was to investigate a potential association between the SLC19A1 80G>A polymorphism and male infertility in a case-control study. The SLC19A1 80G>A polymorphism was determined by means of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assay in 213 infertile Caucasian men and 226 ethnically matched controls. The distribution of SLC19A1 genotypes in the infertile men was as follows: GG 26.8%, GA 51.2%, AA 22.1% and in fertile men: GG 24.8%, GA 50.4%, AA 24.8%, and was comparable in the both the evaluated groups. Odds ratios (95% confidence interval, CI): 0.90 (0.59-1.38) and 0.88 (0.56-1.36) for dominant and recessive models remained non-significant, also after adjustment for age: 0.89 (0.57-1.37) and 0.80 (0.51-1.25), respectively. Our study demonstrated that polymorphism 80G>A of the SLC19A1 gene is not associated with male infertility.

Association of the MDR1 (ABCB1) gene 3435C>T polymorphism with male infertility.

Infertility is a common problem affecting one in six couples, and in 30% of infertile couples, the male factor is a major cause due to defective sperm quality. P-glycoprotein (P-gp), a product of the MDR1 (ABCB1) gene, may be a link between genetic and environmental factors contributing to the development of male infertility because pesticides (P-gp substrates) are well established factors of male infertility. The aim of the present study was to examine the effect of the MDR1 gene 3435C>T polymorphism on male infertility. In total, 162 male patients undergoing semen analysis due to initial infertility workup were included in the study. The control group consisted of 191 healthy males with proven fertility. MDR1 3435C>T genotyping was performed by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Assessment of MDR1 genotypes among the infertile men showed that 17.9% of subjects were carriers of the CC genotype, 58.0% were CT and 24.1% were TT. Among fertile men, 30.4% of subjects were characterised by the CC genotype, 49.7% were CT and 19.9% were TT. In addition, the frequency of carriers of at least one T allele (i.e., CT and TT genotypes) among infertile and fertile subjects was 82.1% and 69.6%, respectively. The risk of infertility was significantly elevated by two-fold in individuals carrying at least one T allele (CT and TT genotypes: p = 0.009, OR = 2.00, 95% CI: 1.20-3.32). Furthermore, this elevated risk was still found when considering each of the CT and TT genotypes alone (TT genotype: p = 0.027, OR = 2.05, 95% CI: 1.09-3.86; CT genotype: p = 0.013, OR = 1.98, 95% CI: 1.16-3.36). This preliminary report suggests that P-gp may play some role in male infertility, mediating detrimental effects of environmental factors.

Comparison of embryological and clinical outcome in GnRH antagonist vs. GnRH agonist protocols for in vitro fertilization in PCOS non-obese patients. A prospective randomized study.

PURPOSE: Embryological and clinical efficacy of gonadotropin-releasing hormone (GnRH) antagonist and agonist stimulation protocols in non-obese women with polycystic ovarian syndrome (PCOS) were compared. METHODS: A prospective randomized study. SETTING: Medical University Hospital. PATIENTS: 70 infertile PCOS patients; 33 in GnRH antagonist and 37 in GnRH agonist group. RESULTS: Similar mature metaphase II oocyte rate (76% vs. 76%) was observed in both protocols. Optimal pronuclear morphology zygotes dominated in both groups (64% vs. 66%). Transferred embryo quality did not differ in both protocols. No significant differences between both protocols were found in delivery rate (p = 0.481), pregnancy rate (p = 0.810), multiple pregnancy rate (p = 0.501), miscarriage rate (p = 0.154), fertilization rate (p = 0.388) and implantation rate (p = 1.000). Duration of stimulation and total follicle-stimulating hormone (FSH) dose were significantly lower in GnRH antagonist protocol (p = 0.0005). CONCLUSIONS: GnRH antagonist and agonist protocols in non-obese PCOS patients yield similar embryological and clinical outcomes. Shorter duration of treatment and lower FSH requirement in GnRH antagonist group may be financially beneficial and therefore attractive for patients.

Isolated severe hydrothorax with respiratory distress as a main manifestation of ovarian hyperstimulation syndrome preceded by respiratory tract infection caused by Haemophilus influenzae.

INTRODUCTION: An isolated unilateral pleural effusion as the only presentation of ovarian hyperstimulation syndrome (OHSS) is very rare. This case is an unusual presentation of OHSS after a confirmed respiratory tract infection, with no other coexisting risk factors identified for this syndrome. We also imply that the presence of Haemophilus influenzae in bronchial fluid can increase local reaction to vasoactive cytokines. CASE REPORT: A 32-year-old woman presented at the Department of Reproductive Medicine and Gynaecology of the Pomeranian Medical University after 10 years of infertility with diagnosed hyperprolactinemia followed by bromocriptine treatment. The patient had three IUIs but no pregnancy was achieved. Therefore, ICSI was proposed. After an ovarian hyperstimulation, oocyte aspiration gave 8 oocytes. Although ICSI was performed in all of the oocytes there were 3 fertilizations. The ET of 3 embryos was carried out following 3 days of culture. Three weeks before the gonadotropin administration and a week before GnRH administration the patient had a respiratory tract infection with the most typical syndromes. The infection was treated successfully with over-the-counter medications and antibiotic. Three days after ET the patient was admitted to the ICU with signs of severe dyspnoea. The chest X-ray showed a large pleural effusion over the right lung. Upon admission, thoracocentesis was preformed and 1600 ml of clear fluid was aspirated. The bronchial aspirate showed evidence of Haemophilus influenzae and leukocytes. After three days of standard treatment the chest X-ray revealed no pathology. The patient was discharged asymptomatic on the 4th day of treatment. Serum beta-hCG level was negative on day 12 after ET. CONCLUSIONS: This case suggest that respiratory tract infection prior to stimulation may constitute a new independent risk factor for OHSS. However, the true relation between the respiratory tract infection and susceptibility to OHSS still awaits explanation. Recent or existing respiratory tract infection may be a relative contraindication for starting COH.

Biotechnological and clinical outcome of in vitro fertilization in non-obese patients with polycystic ovarian syndrome.

INTRODUCTION: Polycystic ovarian syndrome (PCOS) is a hormonal and metabolic disorder which poses problems with controlled ovarian stimulation (COH). It has been also postulated that PCOS patients have oocytes and embryos with poorer quality which affects IVF results. AIM: To verify IVF outcome in non-obese patients with PCOS. MATERIALS AND METHODS: IVF results of 71 non-obese PCOS patients with 243 non-obese non-POCS patients, regardless of stimulation protocol, from years 2004-2006 were compared. RESULTS: Biotechnological results of PCOS patients in opposition to non-PCOS patients were respectively as follows: higher average number (10.19 vs. 7.61; p=0.001) and percentage (82.34% vs. 76.25%; p=0.025) of retrieved mature M2 oocytes; similar (77.01% vs. 76.75%; p=0.835) fertilization rate with higher average number of embryos (7.633 vs. 5.650 p=0.003); higher average number (4.830 vs. 3.304; p=0.001) and percentage (65.66% vs. 60.57%; p=0.006) of embryos with optimal Z1 and Z2 pronuclei pattern according to Scott; higher average number of class Aembryos (3.57 vs. 2.34; p=0.001). Similar number of embryos were transferred in both groups (2.408 vs. 2.485, p=0.552). Clinical results in PCOS and non-PCOS patients were as follows: similar stimulation duration (10.53 days vs. 10.31 days; p=0.639) with significant less gonadotropin total usage (1866.54 IU vs. 2276.18 IU; p=0.001). Also clinical pregnancy per transfer (57.75% vs. 41.98%; p=0.021) and delivery per transfer (45.07% vs. 32.51%; p=0.066) were more often in PCOS patients with comparable miscarriages (12,68% vs. 6,58%; p=0.131) and ectopic pregnancy (0.00% vs. 2.06%; p=0.591) rates, respectively. CONCLUSION: PCOS in non-obese patients is linked with good biotechnological and clinical IVF outcome.

Immunophenotypic profiles of peripheral blood lymphocytes on the day of embryo transfer in women undergoing in vitro fertilization.

UNLABELLED: Evaluation of different types of lymphocyte subpopulations in the peripheral blood has unknown and controversial significance in diagnosis of infertility. The aim of the study was to evaluate selected blood lymphocytes in patients treated with intracytoplasmic sperm injection (ICSI). MATERIALS AND METHODS: women were divided into three groups: (1) control fertile group (n=18), (2) infertile women that achieved (n=32), and (3) did not achieve a pregnancy after ICSI (n=26). The following types of leukocytes were analyzed by three-colour flow cytometry by detection of specific CD antigens: lymphocytes T (CD3+), B (CD19+ and CD5+CD19+), T and B (CD5+), NK cells (CD56+CD16-, CD56-CD16+, CD56+CD16+, CD56brightCD16-, CD56dimCD16+). Additionally, the antigen of early activation (CD69) was evaluated on T, B and NK cells. The results were presented as a percentage and total counts of all lymphocytes. RESULTS: The percentage of total NK cells (CD56+CD16+, CD56+CD16- and CD56-CD16+) did not differ between pregnant and non pregnant women and was lower comparing to control group. Fractions of CD56-CD16+ cells were higher in pregnant vs. non-pregnant women. The percentages of CD56brightCD16- NK cells were higher in control group comparing to both ICSI treated groups. Other fractions of lymphocyte subpopulations, including activated cells (with CD69 expression) did not differ between the analyzed groups. Total counts of CD56-CD16+ cells were higher in pregnant vs. non-pregnant group, and the CD56brightCD16- cells was more abundant in control group vs. women with unsuccessful ICSI. CONCLUSIONS: Testing of peripheral blood NK cells and the others lymphocytes has limited value as a prognostic factor in ICSI treated patients. The antigen of early lymphocytic activation (CD69) has not any predictive value in prognosis of ICSI outcome.

Growth factors effects on preimplantation development of mouse embryos exposed to tumor necrosis factor alpha.

The success rates of assisted reproduction techniques are still unsatisfactory. Relatively few in vitro cultured embryos reach the blastocyst stage. The purpose of the study was to evaluate the protective potential of epidermal growth factor (EGF), insulin-like growth factors 1 and 2 (IGF-I, IGF-II) and stem cell factor (SCF) on in vitro development of pre-implantation mouse embryos exposed to tumor necrosis factor alpha (TNFalpha). C3B6F1 female mice were superovulated with 5 IU of pregnant mare serum gonadotropin (PMSG) and 48 h later with 5IU of equine chorionic gonadotropin (eCG). Following the second injection females were mated with DBA males. Two cell embryos were flushed out from the fallopian tubes 40 h after eCG administration. After retrieval, the embryos were divided into control and experimental media and incubated in groups of ten for 96 h (37 degrees C, 5%CO(2), in droplets of 50 microl under mineral oil). In the first part of experiment, the embryo development was tested in media containing EGF, IGF-I, IGF-II, SCF, TNF-alpha (1 to 1000 ng/ml). In the second part of the study, the development of embryos was examined in medium containing 100 ng/ml TNFalpha and one of following factors: IGF-I, IGF-II; EGF or SCF (100 ng/ml). During the culture embryos were examined at 24 hours intervals to assess the embryo development. Blastocyst rate was determined following 96 hours of culture. Evaluation of total blastocyst cell number (TB) and inner cell mass (ICM) was also performed. TNFalpha significantly reduced (p<0.05) the blastocyst rates as well as TB and ICM. The examined growth factors improved the development of embryos exposed to TNFalpha. Thus, in this study, the protective action of IGF-I and II, EGF and SCF against the detrimental influence of TNFalpha was demonstrated.

Growth factors protect in vitro cultured embryos from the consequences of oxidative stress.

The aim of the study was to evaluate the effect of insulin-like growth factors (IGF1 and IGF2), stem cell factor (SCF) and epidermal growth factor (EGF) on the development of embryos exposed to oxidative stress. C3B6F1 female mice were stimulated with 5 IU of pregnant mare serum gonadotropin and 5 IU of equine chorionic gonadotropin (eCG). Two-cell embryos were flushed out from the fallopian tubes 40 h after eCG administration and mating with DBA males. In each experiment embryos were divided into three groups and cultured in (1) control medium, (2) control medium with 0.1 mM hydrogen peroxide and (3) control medium with hydrogen peroxide and separately with IGF1, IGF2, SCF or EGF in concentrations of 1 ng/ml, 10 ng/ml and 100 ng/ml. Under phase-contrast microscopy, 8-cell and compacted embryos, and early, expanded, hatched and outgrown blastocysts were counted at 24 h. The total blastocyst (TB) and inner cell mass (ICM) cell numbers were established by differential staining. Blastocyst cell viability was examined under fluorescence microscopy. To detect apoptosis, TUNEL was performed and visualized under a laser scanning confocal microscope. Hydrogen peroxide decreased embryo growth, blastocyst rates, blastocyst cell viability as well as TB and ICM counts. The TUNEL reaction revealed significantly more apoptotic cells in oxidative stress conditions. Tested factors revealed a varying extent of protective activity against oxidative stress caused by hydrogen peroxide. In media containing hydrogen peroxide and one of the four tested factors (IGF1, IGF2, SCF or EGF) the embryos developed faster than in media with hydrogen peroxide alone. IGF1, IGF2 and EGF increased both TB and (or) ICM counts in embryos exposed to hydrogen peroxide. All tested factors reduced the number of apoptotic cells (TUNEL) in embryos exposed to hydrogen peroxide.

Methods of embryo scoring in in vitro fertilization.

It has been 25 years since the introduction of in vitro fertilization (IVF) for treatment of infertility. During this time very dynamic advances have taken place in all aspects of assisted reproductive technology (ART). The rapid improvement in embryological methods, especially these related to preimplantation embryo evaluation are of great importance. This article is a review of embryo classification systems utilized in ART programs. The most widely used scoring systems of zygotes and embryos (including blastocysts) are described. Additionally, the advantages of advanced embryo classifications in relation to ART success rates are presented.

Evaluation of mouse preimplantation embryos exposed to oxidative stress cultured with insulin-like growth factor I and II, epidermal growth factor, insulin, transferrin and selenium.

Blastocyst culture requires strictly defined culture media to sustain its viability and quality. Although blastocyst media are commercially available, they do not meet all the needs and research focused on blastocyst-promoting agents is on the way. The aims of the study were to evaluate the significance of insulin-like growth factors I (IGF-I) and II (IGF-II); epidermal growth factor (EGF) and a mixture of insulin, transferrin and selenium (ITS) on the development of embryos exposed to oxidative stress. C3B6F1 mice were stimulated with 5 IU of pregnant mare serum gonadotropin following by administration of 5 IU of equine chorionic gonadotropin and mating with DBA males. The mice were killed 40 h after eCG injection by cervical dislocation and then the 2 cell embryos were flushed out from the fallopian tubes. To evaluate whether the growth factors may compensate the unfavorable--oxidative milieu created by hydrogen peroxide (H2O2), the embryos were transferred to 1/ control medium, 2/ control medium+0.1 mM (H2O2) or 3/ control medium+H2O2 enriched with 10(-7) g/ml of IGF-I, IGF-II, EGF or a mixture of insulin (5x10(-6) g/ml), transferrin (5 x10(-6) g/ml) and selenium (5x10(-9) g/ml; ITS). Embryos were evaluated 96-144 hours following eCG injection. In the study the dynamics of embryo development and blastocyst cell numbers (including inner cell mass) were assessed. The morphological evaluation comprised viability and apoptosis (TUNEL). In oxidative stress setting, IGF-I, IGF-II, EGF and ITS minimized the negative influence of H2O2, and embryos developed faster than in control conditions. Blastocysts cultured with hydrogen peroxide and growth factors or ITS displayed normal morphology and had more cells--also within the inner cell mass--than those treated only with H2O2. The positive TUNEL reactions were sporadically observed in embryos cultured with hydrogen peroxide supplemented with growth factors. IGF-I, IGF-II, EGF and ITS have a positive effect on pre-implantation embryo development in detrimental culture conditions of oxidative stress.